We have examined the mechanism of E. coli DNA polymerase 1 large fragment using simplified reaction systems containing either (dT)800 or (dA)800 as template. This DNA polymerase was capable of highly processive DNA synthesis, incorporating several hundred dNMP residues per cycle of binding to the template, incorporation, and termination. We found that termination at any given product chain-length was markedly increased by the presence of higher concentrations of the template primer complex. This and results of steady-state kinetic and enzyme/template binding studies suggested that the polymerase has more than one functional site for interaction with polynucleotides. Tryptic peptide mapping of putative DNA polymerase alpha subunits indicated that there is sequence homology between higher Mr (110,00) and lower Mr (50,000-65,000) polypeptides found in homogeneous preparations of the enzyme from calf thymus. A number of hybridoma clonal lines have been generated that appear to be producing monoclonal antibody to calf thymus alpha-polymerase.